After CDDP administration, DNA damage was observed mainly in the S phase. It is reasonable to assume that the DNA was structurally altered by CDDP, leading to DNA replication fork arrest and ultimately resulting in apoptosis. This result was consistent with a known pharmacological effect of CDDP [
12]. In RMG-I, apoptotic cells were minimally increased in the S phase, moreover the cells showing arrest in the G1 and G2/M phases without DNA damage were increased as compared with OVISE. Therefore, the results of the present study support the clinical experience that RMG-I is CDDP-resistant [
11,
13]. After CBDCA administration, DNA damage was seen in the S and G2/M phases in both cell lines. OVISE contained a remarkable cell population rescued from apoptosis and surviving with DNA damage. On the other hand, most RMG-I cells with DNA damage underwent apoptosis. These results suggest that cell lines respond differently to platinum agents, i.e., RMG-I was CDDP-resistant but responded to CBDCA. PTX directly induced apoptosis in M-phase cells but not via DNA damage, an observation consistent with a known pharmacological effect of PTX, i.e. microtubule inhibition [
14]. PTX was confirmed to induce apoptosis through a p53-independent pathway; it was, therefore, expected to have an effect on CCC, in which the p53 mutation is rare [
15,
16]. The mechanism underlying the antitumor effect of PTX is G2/M arrest. Therefore, the combination with CBDCA, an agent inducing DNA damage, in G2/M-phase arrested cells might be effective, at least theoretically. As shown in this study, it is noteworthy that sensitivities to CDDP and CBDCA differed between the CCC cell lines. In practice, CCC is less sensitive to CBDCA/PTX treatment [
4,
6,
17], which is the standard regimen for ovarian cancer. Since the effect of PTX was independent of both the concentration and the response time, these results raise the possibility that repeated administration of PTX at a low dose increases the antitumor effect more than a single administration. These findings support the results of the JGOG3016, i.e. that weekly CBDCA (AUC6)/PTX (80 mg/m
2, weekly × 3) is more effective than tri-weekly CBDCA (AUC 6)/PTX (175 mg/m
2) treatment [
18].
On the other hand, after administration of SN-38, DNA damage occurred in S-phase cells, followed by apoptosis. This confirmed that SN-38 acts as a type I topoisomerase inhibitor [
19]. Furthermore, it appears that SN-38 had an effect on the cell cycle because S-phase arrest continued for more than 120 hours. It is, therefore, possible that the improved administration method for SN-38 increases its antitumor effect. Cells rescued from apoptosis remained in S phase with DNA damage; consequently, the efficacy of combining SN-38 with CDDP, which induces DNA damage mainly in S-phase cells, was supported.
In conclusion, the present results suggest that an effective treatment for CCC with a slow growth rate and a low ratio of S-phase cells would be a combination of agents arresting the cell cycle, thereby causing accumulation of cells in the S phase or the G2/M phase, and agents specifically inducing DNA damage in S-phase cells. The method used in this study allows immunocytochemical detection of γH2AX, which indicates DNA damage even at very low concentrations and has high sensitivity in comparison with the comet assay. Employing this method, we were able to analyze relationships between anti-tumor effects and cell cycle perturbations. Therefore, γH2AX detection is a promising method of simply and rapidly identifying agents potentially effective against CCC.